Photodynamic therapy (PDT) involves the action of light on a photosensitizer retained in diseased, especially cancerous tissue to produce selective cell/tissue kill. The application to the treatment of cancer and other disease states depends upon the relative selective retention of the agent in a tumor or other cancerous tissue, low systemic toxicity and the ability of the activating light to reach the diseased site. Sensitizers in clinical use include the porphyrin derivatives such as hematoporphyrin and a purified form known as dihematoporphyrin.
The sensitizers are chosen for their low systemic toxicity and are retained in most malignant tissue at levels sufficient to elicit a localized photosensitized reaction when activated by light near 630 nm (about 600 nm to about 800 nm). In many cases, the tissues surrounding or overlying the tumors have significantly lower levels of drug allowing for a relative selectivity in the destruction of the malignant tissue.
Porphyrins are naturally occuring compounds which, upon activation by light generate singlet oxygen and possibly superoxide and hydroxyl radicals. This light activation and the generation of reactive chemistry is believed to be responsible for the anti-tumor activity that many of these agents possess. Porphyrins are preferentially accumulated by tumor cells and provide the basis for PDT. This anticancer treatment involves parental, oral or topical administration of the porphyrin and, following accumulation by the tumor, its activation by a laser light directed onto the tumor via a flexible fibre optic tube (See, for example, Dougherty, T. J. In Method in Porphyrin Photosensitization, Edited by D. Kessel, pp. 313-328, 1985 by Plenum Press, New York). Extensive clinical studies with hematoporphyrin derivative (HpD) and Photofrin.RTM. (a mixture of dihematoporphyrin ethers/esters) have been documented in the literature with good success (See, for example, Dougherty, T. J., Photochem. Photobiol., 46, 569 (1987); Kessel, et al., Photochem. Photobiol., 46 563 (1987); Dougherty, T. J., Semin. Surg. Oncol., 2, 24 (1986); McCaughan, J. S., Photochem. Phtobiol. 46, 903 (1987); and Gomer, C. J., Semin. Hematol., 26(1) 27 (1989)). This treatment however, is generally limited to light accessible carcinomas such as carcinomas of the lung, bladder, skin, etc.
Most clinical studies involving PDT have utilized hematoporphyrin derivative (HPD) which consists of a mixture of porphyrin compounds (See, Dougherty, T. J., Photochem. Photobiol. 46, 569 (1987). However, the therapeutic use of HPD is subject to two major limitations. First, at the wavelength of light which activates the compound, the light only poorly penetrates the tissue (See Doiron, et al., pp. 281-291 In:Porphyrins in Tumor Phototherapy, Edited by A. Andreoni and R. Cuulbreddu, Plenum Press, New York, 1984). This effectively limits the size of the tumors that can be treated. Second, HPD tends to accumulate in the skin of patients rendering the patients photosensitive. In most instances, patients must avoid sunlight for up to 6 weeks.
At present the most widely studied compounds for use in PDT are certain dyes, for example, porphyrins and structurally related compounds such as the chlorins, chlorophylls and purpurins, which occur naturally in animals and plants, and phthalocyananines which are synthetic molecules. A few other structurally distinct comounds have also been studied. All of the compounds which have been investigated for use in PDT have the same characteristics in common; i.e., they are pure compounds having well defined structures and absorb light in the range of 600 to 800 nm.
Benzoporphyrin derivative (BPD) represents a second generation of photosensitizers which are superior to HPD. BDP is a chlorin-like porphyrin composed of four structural analogues following synthesis. All four analogues have an identical reduced tetrapyrrol porphyrin ring. Each analogue differs only by the position of a cyclohexadiene ring which may be fused either at ring A or ring B of the porphyrin (A or B analogues) and the presence of either two acid groups (diacids) or one acid and one ester group (monoacids) at rings C and D of the porphyrin (See FIG. 1). All four analogues are hydrophobic, absorb red light at about 700 nm and efficiently produce singlet oxygen. Despite the sensitivity of all four molecules, they differ in their light activated cytoxicity in vitro and in vivo.
Two clear advantages are presented by BPD. The first is that the compound is activated by light of a longer wavelength than activates HPD. The longer wavelength light can penetrate deeper into tissue. In addition, its properties should markedly reduce patient photosensitivity.
BPD, however, suffers from the drawback that it is not water-soluble and its pharmaceutical application requires the development of a suitable delivery vehicle for parenteral administration.
Liposomes are completely closed structures comprising lipid bilayer membranes containing an encapsulated aqueous volume. Liposomes may contain many concentric lipid bilayers separated by aqueous channels (multilamellar vesicles or MLVs), or alternatively, they may comprise a single membrane bilayer (unilamellar vesicles). The lipid bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. In the membrane bilayer, the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer, whereas the hydrophilic (polar) "heads" orient toward the aqueous phase. The basic structure of liposomes may be made by a variety of techniques known in the art.
Liposomes have typically been prepared using the process of Bangham et al., (1965 J. Mol. Biol., 13: 238-252), whereby lipids suspended in organic solvent are evaporated under reduced pressure to a dry film in a reaction vessel. An appropriate amount of aqueous phase is then added to the vessel and the mixture agitated. The mixture is then allowed to stand, essentially undisturbed for a time sufficient for the multilamellar vesicles to form. The aqueous phase entrapped within the liposomes may contain bioactive agents, for example drugs, hormones, proteins, dyes, vitamins, or imaging agents, among others.
Liposomes may be reproducibly prepared using a number of currently available techniques. The types of liposomes which may be produced using a number of these techniques include small unilamellar vesicles (SUVs) [See Papahadjapoulous and Miller, Biochem. Biophys. Acta., 135, p. 624-638 (1967)], reverse-phase evaporation vesicles (REV) [See U.S. Pat. No. 4,235,871 issued Nov. 25, 1980], stable plurilamellar vesicles (SPLV) [See U.S. Pat. No. 4,522,803, issued Jun. 11, 1985], and large unilamellar vesicles produced by an extrusion technique as described in U.S. patent application Ser. No. 310,495 filed Feb. 13, 1989, Cullis et. al., entitled "Extrusion Technique for Producing Unilamellar Vesicles", relevant portions of which are incorporated herein by reference.
Another class of multilamellar liposomes are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in cullis et al., U.S. patent application Ser. No. 122,613, filed Nov. 17, 1987, U.S. Pat. No. 4,975,282 issued Dec. 4, 1990 PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies". U.S. Pat. No. 4,721,612 to Janoff et al. describes steroidal liposomes for a variety of uses. The teachings of these references as to preparation and use of liposomes as well as other relevant sections are incorporated herein by reference.